Folate-sensitive fragile sites are nonrandom regions of the human genome that exhibit excess fragility when cells are grown under conditions of pyrimidine nucleotide stress. To date, 15 rare heritable folate-sensitive fragile sites have been destroyed as well as a large number of folate-sensitive common sites which appear to be virtually ubiguitous in the population. There has been great interest in the folate-sensitive fragile sites because one of them, the rare heritable site at Xq27, is associated with the Fragile X Syndrome, the most common inherited cause of mental retardation. The Xq27 fragile site and the fragile X gene mutation map to the same position on the X chromosome and therefore may be identical. In males, and usually in females, there is a direct relationship between expression of the fragile site and manifestation of the syndrome. However, fragile site expression has not been demonstrated in lymphocytes in virtually all phenotypically normal homozygotes and approximately 60% of phenotypically normal homozygotes. a simple and efficient method of identifying these nonpenetrant individuals would be extremely useful in genetic counseling situations and linkage analysis. The relationship between the Xq27 fragile site, the rare autosomal fragile sites, and the common fragile sites is not known. Although all of the sites appear to be associated with errors in DNA replication and/or repair, methods that induce or enhance one group of fragile sites may have no effect on another. The proposed research will compare the effect of different inhibitors of DNA replication and repair on the expression of common fragile sites, rare autosomal fragile sites, and the rare fragile site at Xq27. This comparison will provide data useful in: (1) formulating models of mechanism(s) of expression of folate-sensitive fragile sites; and (2) developing an efficient method of identifying phenotypically normal nonexpressing carriers of the fragile X gene.